067 | Redefining amphibian innate immunity: first in vitro and in vivo evidence of phagocytic function in blood cells

Valbona Aliko¹, Erion Sukaj², Cristiana Roberta Multisanti³, Federica Impelliteri⁴, Caterina Faggio⁴ | ¹University of Tirana, Faculty of Natural Sciences, Department of Biology, Tirana, Albania; ²Western Balkans University, Faculty of Medicine and Technical Medical Sciences, Tirana, Albania; ³Department of Veterinary Sciences, University of Messina, Italy; ⁴University of Messina, Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, Messina, Italy.

Phagocytosis is a cornerstone of innate immunity, yet in amphibians it remains largely unexplored due to the lack of standardized methodologies and quantitative indices. To date, no study has combined in vitro and in vivo approaches to characterize phagocytic function in amphibian blood cells. Here, we present the first integrated in vitro and in vivo analysis of phagocytosis in amphibians, providing novel mechanistic insights into cellular immune responses. Phagocytosis was induced using Saccharomyces cerevisiae in frog blood cells, in the absence and presence of zinc oxide nanoparticles (ZnO-NPs). In vitro assays involved controlled incubation, fixation, Giemsa staining, and digital microscopy. In vivo phagocytosis was assessed by intraperitoneal injection of neutral red–labeled yeast, enabling real-time tracking of antigen uptake. Phagocytic capacity (CP) and phagocytic index (PI) were quantified through direct microscopic evaluation. Both models revealed rapid immune activation, with peak phagocytic activity occurring between 1.5 and 2.5 hours post-stimulation. ZnO-NPs significantly enhanced phagocytic responses, increasing both the number of engaged cells and intracellular antigen load. Mechanistically, phagocytosis was associated with increased cell motility, vesicle formation, lysosomal activation, and cytoplasmic remodeling. Unexpectedly, nucleated erythrocytes actively participated in antigen adhesion and internalization, forming pseudopod-like extensions and enclosing yeast particles. Morphometric analysis confirmed significant increases in cell surface area and integrated density during erythrocyte-mediated phagocytosis. These findings redefine the immunological role of amphibian blood cells and establish a robust framework for comparative innate immunity studies. This study provides the first in vitro and in vivo evidence of phagocytic function in amphibian blood cells, revealing an unexpected immune role for nucleated erythrocytes and redefining amphibians as powerful models for investigating the evolution of innate immunity.